CytoSelect 96- Well Cell Migration and Invasion Assay (8 µm, Fluorometric Format)

Transcription

1 Product Manual CytoSelect 96- Well Cell Migration and Invasion Assay (8 µm, Fluorometric Format) Catalog Number CBA C 2 x 96 assays (96 migration + 96 invasion) FOR RESEARCH USE ONLY Not for use in diagnostic procedures

2 Introduction Cell migration is a highly integrated, multistep process that orchestrates embryonic morphogenesis, tissue repair and regeneration. It plays a pivotal role in the disease progression of cancer, mental retardation, atherosclerosis, and arthritis. The initial response of a cell to a migration-promoting agent is to polarize and extend protrusions in the direction of the attractant; these protrusions can consist of large, broad lamellipodia or spike-like filopodia. In either case, these protrusions are driven by actin polymerization and can be stabilized by extracellular matrix (ECM) adhesion or cell-cell interactions. The ability of malignant tumor cells to invade normal surrounding tissue contributes in large part to the significant morbidity and mortality of cancers. Invasiveness requires several distinct cellular functions including adhesion, motility, detachment, and extracellular matrix proteolysis. Metastatic cells produce many proteolytic enzymes (e.g. lysosomal hydrolysates, collagenases, plasminogen activators) while the expression of certain cell surface protease receptors is also increased. Cell Biolabs CytoSelect 96-well Cell Migration and Invasion Assay Kit utilizes a polycarbonate membrane plate (8 µm pore size) or basement membrane-coated inserts to assay the migratory or invasive properties of cells. The kit does not require you to prelabel the cells with Calcein AM or remove non-migratory or non-invasive cells (i.e. cotton swabbing). Any migratory or invasive cells are first dissociated from the membrane, then lysed and detected with CyQuant GR Dye. The CytoSelect 96-well Cell Migration and Invasion Assay Kit provides a robust system for the quantitative determination of cell migration. Each assay contains sufficient reagents for the evaluation of 96 samples. The 8 µm pore size is optimal for epithelial and fibroblast cell migration. However, in the case of leukocyte chemotaxis, a smaller pore size (3 µm) is recommended. Related Products 1. CBA-100: CytoSelect 24-Well Cell Migration Assay (8µm, Colorimetric) 2. CBA-101: CytoSelect 24-Well Cell Migration Assay (8µm, Fluorometric) 3. CBA-101-C: CytoSelect 24-Well Cell Migration and Invasion Assay (8µm, Fluorometric) 4. CBA-102: CytoSelect 24-Well Cell Migration Assay (5µm, Fluorometric) 5. CBA-103: CytoSelect 24-Well Cell Migration Assay (3µm, Fluorometric) 6. CBA-104: CytoSelect 96-Well Cell Migration Assay (3µm, Fluorometric) 7. CBA-105: CytoSelect 96-Well Cell Migration Assay (5µm, Fluorometric) 8. CBA-106: CytoSelect 96-Well Cell Migration Assay (8µm, Fluorometric) 9. CBA-112: CytoSelect 96-Well Cell Invasion Assay (Basement Membrane, Fluorometric) 10. CBA-120: CytoSelect 24-Well Wound Healing Assay (Light Microscopy) 11. CBA-125: Radius 24-Well Cell Migration Assay (Microscopy) 12. CBA-130: CytoSelect 96-Well Cell Transformation Assay (Soft Agar Colony Formation) 2

3 Kit Components well Cell Migration Plate (Part No ): One sterile 96-well plate (see Figure 1 for components) 96-well Cell Invasion Plate (Part No ): One sterile 96-well plate containing ECM-coated inserts (see Figure 1 for components) 96-well Cell Harvesting Tray (Part No ): Two 96-well trays Cell Detachment Solution (Part No ): Two 20 ml bottles 4X Lysis Buffer (Part No ): Two 10 ml bottles CyQuant GR Dye (Part No ): Two 75 µl tubes Top Plate Cover Middle Migration Plate Bottom Membrane Chamber Feeder Tray Figure 1: Components of the 96-well Cell Migration Plate. Materials Not Supplied 1. Migratory cell lines 2. Cell culture medium 3

4 3. Serum free medium, such as DMEM containing 0.5% BSA, 2 mm CaCl 2 and 2 mm MgCl 2 4. FBS or desired chemoattractant 5. Cell culture incubator (37ºC, 5% CO 2 atmosphere) 6. Light microscope well plate suitable for a fluorescence plate reader 8. Fluorescence plate reader Storage Store all components at 4ºC. Cell Migration Assay Principle The CytoSelect 96-well Cell Migration Assay Kit contains a polycarbonate membrane chamber (8 µm pore size) in a 96-well plate. The membrane serves as a barrier to discriminate migratory cells from non-migratory cells. Migratory cells are able to extend protrusions towards chemoattractants (via actin cytoskeleton reorganization) and ultimately pass through the pores of the polycarbonate membrane. These migratory cells are then dissociated from the membrane and subsequently detected with CyQuant GR Dye. 4

5 Cell Migration Assay Protocol 1. Allow the 96-well Migration Plate to warm up at room temperature for 10 minutes. 2. Prepare a cell suspension containing x 10 6 cells/ml in serum free media. Agents that inhibit or stimulate cell migration can be added directly to the cell suspension. Note: Overnight starvation may be performed prior to running the assay 3. Under sterile conditions, separate the cover and membrane chamber from the 96-well Migration Plate. 4. Add 150 µl of media containing 10% fetal bovine serum or desired chemoattractant(s) to the wells of the feeder tray. 5. Place the membrane chamber back into the feeder tray (containing chemoattractant solution). Ensure no bubbles are trapped under the membrane. 6. Gently mix the cell suspension (without chemoattractant) from step 2 and add 100 µl to the membrane chamber. 7. Finally, cover the plate and transfer to a cell culture incubator for 2-24 hours. 8. Just prior to the end of the incubation, pipette 150 µl of prewarmed Cell Detachment Solution into wells of the clean, 96-Well Cell Harvesting Tray (provided). 9. Carefully remove the 96-well Migration Plate from the incubator. Separate the membrane chamber from the feeder tray. 10. Remove the cells/media from the top side of the membrane chamber by aspirating or inverting. Place the membrane chamber into the Cell Harvesting Tray containing 150 µl of Cell Detachment Solution (step 8). Incubate 30 minutes at 37ºC. 11. Completely dislodge the cells from the underside of the membrane by gently tilting the membrane chamber several times in the Cell Detachment Solution. 12. Prepare sufficient 4X Lysis Buffer/CyQuant GR dye solution for all samples by diluting the dye 1:75 in 4X Lysis Buffer (for example, add 5 µl dye to 370 µl of 4X Lysis Buffer). 13. Add 50 µl of 4X Lysis Buffer/CyQuant GR dye solution to each well (already containing 150 µl of Cell Detachment Solution). Incubate 20 minutes at room temperature. 14. Transfer 150 µl of the mixture to a 96-well plate suitable for fluorescence measurement. Read the fluorescence with a fluorescence plate reader at 480 nm/520 nm. Cell Invasion Assay Principle The CytoSelect 96-well Cell Invasion Assay Kit contains polycarbonate membrane inserts (8 µm pore size) in a 96-well plate. The upper surface of the insert membrane is coated with a uniform layer of dried basement membrane matrix solution. This basement membrane layer serves as a barrier to discriminate invasive cells from non-invasive cells. Invasive cells are able to degrade the matrix proteins in the layer, and ultimately pass through the pores of the polycarbonate membrane. Finally, the invaded cells are dissociated from the membrane and subsequently detected with CyQuant GR Dye. 5

6 Cell Invasion Assay Protocol 1. Under sterile conditions, allow the invasion plate to warm up at room temperature for 10 minutes. 2. Rehydrate the basement membrane layer of the membrane inserts by adding 100 µl of warm, serum-free media to the inner compartment. Incubate at room temperature for 1 hour. 3. Prepare a cell suspension containing x 10 6 cells/ml in serum free media. Agents that inhibit or stimulate cell invasion can be added directly to the cell suspension. 4. Carefully remove the rehydration medium (step 2) from the inserts without disturbing the basement membrane layer. Note: It will not affect the assay performance if a small amount of rehydration medium is left in the compartment. 5. Under sterile conditions, separate the cover and membrane chamber from the feeder tray. Add 150 µl of media containing 10% fetal bovine serum or desired chemoattractant(s) to the wells of the feeder tray. 6. Place the membrane chamber back into the feeder tray (containing chemoattractant solution). Ensure no bubbles are trapped under the membrane. 7. Gently mix the cell suspension from step 3 and add 100 µl to the membrane chamber. 6

7 8. Finally, cover the plate and transfer to a cell culture incubator for hours. 9. Just prior to the end of the incubation, pipette 150 µl of prewarmed Cell Detachment Solution into wells of the clean, 96-Well Cell Harvesting Tray (provided). 10. Carefully remove the 96-well Invasion Plate from the incubator. Separate the membrane chamber from the feeder tray. 11. Remove the cells/media from the top side of the membrane chamber by aspirating or inverting. Place the membrane chamber into the Cell Harvesting Tray containing 150 µl of Cell Detachment Solution (step 9). Incubate 30 minutes at 37ºC. 12. Completely dislodge the cells from the underside of the membrane by gently tilting the membrane chamber several times in the Cell Detachment Solution. 13. Prepare sufficient 4X Lysis Buffer/CyQuant GR dye solution for all samples by diluting the dye 1:75 in 4X Lysis Buffer (for example, add 5 µl dye to 370 µl of 4X Lysis Buffer). 14. Add 50 µl of 4X Lysis Buffer/CyQuant GR dye solution to each well (already containing 150 µl of Cell Detachment Solution). Incubate 20 minutes at room temperature. 15. Transfer 150 µl of the mixture to a 96-well plate suitable for fluorescence measurement. Read the fluorescence with a fluorescence plate reader at 480 nm/520 nm. Example of Results The following figures demonstrate typical with the CytoSelect Cell Migration and Invasion Assay Kit. Fluorescence measurement was performed on SpectraMax Gemini XS Fluorometer (Molecular Devices) with a 485/538 nm filter set and 530 nm cutoff. One should use the data below for reference only. This data should not be used to interpret actual results RFU Cell Number Figure 2: Quantitation of Human HT HT-1080 cells were titrated in Cell Detachment Buffer, then subsequently lysed and detected with 4X Lysis Buffer/Cyquant GR Dye (150 µl cell suspension was mixed with 50 µl of 4X Lysis Buffer/dye). 7

8 RFU Cytochalasin D 0.1% FBS 1% FBS 10% FBS 10% FBS + Cytochalasin D Figure 3: HT-1080 Chemotaxis. HT-1080 cells were allowed to migrate toward FBS for 4 hrs in the presence or absence of 2 µm Cytochalasin D, 30,000 cells were used in each assay. Migratory cells on the bottom of the polycarbonate membrane were detached and quantified by CyQuant GR Dye as described in the Assay Protocol RFU NIH3T3 NIH-3T3+ Cytochalasin D HT-1080 HT Cytochalasin D Figure 4: HT-1080 Invasion Assay. HT-1080 or NIH3T3 cells were allowed to invade toward 10% FBS for 24 hrs in the presence or absence of 2 µm Cytochalasin D, 200,000 cells were used in each assay. Invaded cells on the bottom of the polycarbonate membrane were stained (top) and quantified by CyQuant GR Dye as described in Assay Protocol. 8

9 References 1. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR. (2003) Science 302, Horwitz R, Webb D. (2003) Curr Biol. 13, R Lauffenburger DA, Horwitz AF. (1996) Cell 84, Recent Product Citations 1. Sharma, N. L. et al. (2014). The ETS family member GABPα modulates androgen receptor signalling and mediates an aggressive phenotype in prostate cancer. Nucleic Acids Res. 42: Zecchini, V. et al. (2014). Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer. EMBO J. 33: Ardiani, A. et al. (2014). Vaccine-mediated immunotherapy directed against a transcription factor driving the metastatic process. Cancer Res. 74: Sharma, N.L. et al. (2014). The ETS family member GABPa modulates androgen receptor signaling and mediates an aggressive phenotype in prostate cancer. Nucleic Acids Res /nar/gku Axlund, S.D. et al. (2010). HOXC8 inhibits androgen receptor signaling in human prostate cancer cells by inhibiting SRC-3 recruitment to direct androgen target genes. Mol. Cancer Res. 8: Alfano, R.W. et al. (2009). Matrix metalloproteinase-activated anthrax lethal toxin inhibits endothelial invasion and neovasculature formation during in vitro morphogenesis. Mol. Cancer Res. 7: Eckstein, N. et al. (2008). EGFR-pathway analysis identifies amphiregulin as a key factor for cisplatin resistance of human breast cancer cells. J. Biol. Chem. 283: License Information This product is provided under an intellectual property license from Life Technologies Corporation. The purchase of this product conveys to the buyer the non-transferable right to use the purchased product and components of the product only in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The sale of this product is expressly conditioned on the buyer not using the product or its components, or any materials made using the product or its components, in any activity to generate revenue, which may include, but is not limited to use of the product or its components: (i) in manufacturing; (ii) to provide a service, information, or data in return for payment; (iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they are resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad CA USA or outlicensing@lifetech.com. 9

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